This invention relates generally to proteins, functionally equivalent pharmacophores and methods of creating and/or detecting inhibitors of prion formation. Specifically, the invention relates to small molecules, peptides and peptide analogs with the ability to either inhibit prion formation or replication and methods of treating a neuropathology such as a prion-mediated neuropathology.
Prions are infectious pathogens that cause central nervous system spongiform encephalopathies in humans and animals. Prions are distinct from bacteria, viruses and viroids. The predominant hypothesis at present is that no nucleic acid component is necessary for infectivity of prion protein. Further, a prion which infects one species of animal (e.g., a human) will not efficiently infect another (e.g., a mouse).
From a clinical perspective, the prion diseases represent a variety of neurodegenerative states characterized at the neuropathologic level by the presence of spongiform degeneration and astrocytic gliosis in the central nervous system (DeArmond and Prusiner (1996) Current Topics in MicroBiology and Immunology, 207:125-146). Frequently, protein aggregates and amyloid plaques are seen that are often resistant to proteolytic degradation. The neuroanatomic distribution of the lesions varies with the specific types of prion disease. In humans, sporadic Creutzfeldt-Jakob Disease (CJD) accounts for 85% of all cases. The disease presents in the sixth decade of life with dementia and ataxia. Familial disease carries a variety of monikers such as Gertsmann-Straussler-Scheinker disease (GSS), familial CJD (fCJD) and Fatal Familial Insomnia (FFI) that relate the precise mutation in the PrP gene to a clinical syndrome (Prusiner and Hsaio (1994) Annals of Neurology, 35:385-395; Parchi, et al. (1996) Annals of Neurology, 39:767-778; Montagna, et al. (1998) Brain Pathology, 8:515-520). Disease typically presents in the fourth decade of life with an autosomal dominant pedigree. While the infectious prion diseases represents less than 1% of all cases, their link to mad cow disease in the U.K. (new variant CJD), growth hormone inoculations in the U.S. and France (iatrogenic CJD), and ritualistic cannibalism in the Fore tribespeople (Kuru) have raised the public awareness of this facet of the disease (Devillemeur, et al. (1996) Neurology, 47:690-695; Hill, et al. (1997) Nature, 389:448-450; Goodfield (1997) Nature, 387:841-841).
A critical advance in our understanding of prion diseases came with the partial purification of a proteinaceous material that retained the ability to reinfect laboratory rodents (McKinley, et al. (1983) Cell, 35:57-62). Micro-sequencing and molecular biologic tools led to the cloning of the prion gene, a normal component of mammalian and avian genomes (Prusiner, et al. (1984) Cell, 38:127-134). The gene contains a single open reading frame and codes for a protein that is proteolytically processed and glycosylated to form a macromolecule with 219 amino acids, a disulfide bridge, two N-linked sugars and a glycophosphotidyl inositol anchor that is exported to the cell surface and concentrated in an endocytic compartment known as the caveolar space (Endo, et al. Biochemistry, 28:8380-8 1989); Stahl, et al. Biochemistry 29:8879-84 (1990); Yost, et al. Nature, 343:669-72 (1990); DeFea, et al. J. Biol. Chem., 269:16810-16820 (1994); Hegde, et al., Science 279:827-34 (1998)). Biophysical characterization of the deglycosylated recombinant PrP refolded into a monomeric form resembling the normal cellular isoform (PrPC) reveals a two domain molecule with an N-terminal region (57-89) that binds 4 Cu++ atoms per chain (Viles, et al. (1999) Proc. Natl. Acad. Sci. USA, 96:2042-2047) and a C-terminal region (124-231) that contains 3 substantial helices and 2-3 residue xcex2-strands joined by 2-3 hydrogen bonds (see FIG. 1) (Riek, et al. (1996) Nature, 382:180-182; James, et al. (1997) Proc. Natl. Acad. Sci. USA, 94:10086-10091; Donne, et al. (1997) Proc. Natl. Acad. Sci. USA, 94:13452-13457). By contrast, the disease causing form of the prion protein (PrPSc) is a multimeric assembly substantially enriched in xcex2-sheet structure (40% xcex2-sheet, 30% xcex1-helices as judged by FTIR spectroscopy) (Pan, et al. (1993) Proc. Natl. Acad. Sci. USA, 90:10962-10966). Immunologic studies of PrPSc suggest that the conformational change is largely in the region from residues 90-145 or perhaps 175 (Peretz, et al. (1997) J. Mol. Biol. 273:614-622) These features have been codified in a model of PrPSc (see FIG. 2) that emphasize the dramatic conformational distinction between PrPC and PrPSc.
A large number of genetic and transgenetic studies have helped to cement the role of the prion protein in the pathogenesis of this group of neurodegenerative diseases. First, a variety of genetic linkage studies of kindreds with familial prion diseases mapped the defect to the Prn-p locus. Subsequent studies identified specific point mutations that caused inherited disease (Hsiao, et al. (1989) Nature, 338:342-345; Dlouhy, et al. (1992) Nat. Genet., 1:64-67; Petersen, et al. (1992) Neurology, 42:1859-1863; Poulter, et al. Brain 115:675-85 (1992); Gabizon, et al. (1993) Am. J. Hum. Genet., 53:828-835). These loci are shown in FIG. 3. Subsequently, the Prn-p gene was knocked out in mice with no obvious phenotypic sequelae (Bxc3xceler, et al. (1992) Nature, 356:577-582). While wild type mice will develop a prion disease xcx9c180d after intracerebral inoculation, the hemizygous animals require xcx9c400d to succumb to an infectious inoculum and the homozygous knockouts are resistant to prion infection (Prusiner, et al. (1993) Proc. Natl. Acad. Sci. USA, 90:10608-10612; Bxc3xceler, et al. (1994) Molecular Medicine, 1:19-30). Transgenic mice carrying a sufficiently high number of copies of mutant gene (the human GSS mutation P101L) on the knockout background develop a spontaneous neurodegenerative disease that is faithful to the neuropathologic expectations developed from a study of the human kindreds. Knockout mice carrying a redacted form of the PrP transgene (90-141; 175-231) also develop a prion disease upon inoculation with full length RML prions (Supattapone, et al. (1999) Cell, 96:869-878). The infection process is more efficient with the xe2x80x9cminixe2x80x9d RML prion demonstrating that an artificial prion can be created and that replication efficiency demands fidelity at the amino acid sequence level.
While the predominantly helical PrPC and xcex2-sheet rich PrPSc have exceptionally different secondary and tertiary structures as judged by CD, FTIR, and NMR spectroscopy (Caughey, et al. (1991) Biochemistry, 30:7672-7680; Pan, et al. (1993) Proc. Natl. Acad. Sci. USA, 90:10962-10966; Riek, et al. (1996) Nature, 382:180-182; James, et al. (1997) Proc. Natl. Acad. Sci. USA, 94:10086-10091; Donne, et al. (1997) Proc. Natl. Acad. Sci. USA, 94:13452-13457), they appear to share a common amino acid sequence and disulfide bridge (Cohen and Prusiner (1998) Annual Review of Biochemistry, 67:793-819). Recent work has shown that a conformational change that is aided by an auxiliary molecule is an obligatory step in PrPSc formation (Telling, et al. (1995) Cell, 83:79-90; Kaneko, et al. (1997) J. Mol. Biol. 270:574-586). The exceptional stability of PrPSc and the marginal stability of PrPC together with a variety of transgenetic and cellular transfection studies have led to the conclusion that PrPC is a kinetically trapped intermediate in the folding of PrPSc (Cohen and Prusiner (1998) Annual Review of Biochemistry, 67:793-819). This kinetic barrier can be reduced by exogenous administration of the PrPSc template, mutations to the wild type (wt) PrP sequence, or stochastic processes resulting in infectious, inherited, or sporadic prion diseases. Epitope mapping and peptide studies suggest that much of this conformational plasticity is localized to the middle third of this 231 residue GPI anchored glycoprotein with a 22 amino acid signal sequence (Peretz, et al. (1997) J. Mol. Biol., 273:614-622).
Peptide fragments derived from regions of the PrP sequence have been studied extensively (Gasset, et al. (1992) Proc. Natl. Acad. Sci. USA, 89:10940-10944; Tagliavini, et al. (1993) Proc. Natl. Acad. Sci. USA, 90:9678-9682; Forloni, et al. (1993) Nature, 362:543-546; Come, et al. (1993) Proc. Natl. Acad. Sci. USA, 90:5959-5963; Zhang, et al. (1995) J. Mol. Biol., 250:514-526; Nguyen, et al. (1995) Biochemistry, 34:4186-4192; Kaneko, et al. (1997) J. Mol. Biol. 270:574-586). In particular, peptides chosen from the region 90-145 are compatible with xcex1-helical, irregularly coiled, and xcex2-sheet rich conformations when characterized under different conditions (Zhang, et al. (1995) J. Mol. Biol., 250:514-526). Furthermore, catalytic amounts of xcex2-sheet rich peptides can facilitate the conformational conversion of peptides with distinct structures into xcex2-sheet rich isoforms (Gasset, et al. (1992) Proc. Natl. Acad. Sci. USA, 89:10940-10944; Nguyen, et al. (1995) Biochemistry, 34:4186-4192).
More than a million cattle infected with bovine spongiform encephalopathy (BSE) have entered the food chain in the U.K., and fears that BSE has been transmitted to man were raised when new variant (CJD) appeared in the U.K. Since it is hard to predict the number of cases of this disease that may arise in the future, initiation of the search for an effective therapy is essential. No systematic drug discovery efforts have been attempted owing to difficulties in developing a robust screening assay. Many isolated observations with potential therapeutic implications have been made. For example, several compounds are known to inhibit PrPSc formation in scrapie-infected neuroblastoma cells such as sulfated glycans and the amyloid stain Congo Red (Caughey and Raymond (1991) J. Biol. Chem., 266:18217-18223). However, these compounds are unable to cross the blood-brain barrier, and therefore have no therapeutic benefit after the infection has reached the central nervous system (Caughey, et al. (1993) J. Virol., 67:6270-6272; Ehlers and Diringer (1984) J. Gen. Virol., 651325-1330; Farquhar and Dickinson (1986) J. Gen. Virol., 67:463-473). Other candidates such as polyene antibiotics (Demaimay, et al. (1997) J. Virol., 71:9685-9589) and anthracyclines (Tagliavini, et al. (1997) Science, 276:1119-1122) have very low therapeutic indices. Tetrapyrroles inhibit PrPSc formation and there is some evidence that they can cross the blood-brain barrier (Caughey, et al. (1998) Proc. Natl. Acad. Sci. USA, 95:12117-12122), but at this time, the mechanism of action and in vivo efficacy of these compounds is unknown.
There is a need in the art for molecules with the ability to prevent and/or halt the progression of prion-mediated disorders.
Molecules are disclosed that interact with the cellular components involved in conversion of PrPC to PrPSc. The molecules disclosed can be small molecules, peptides or protein analogs, e.g. analogs of PrPC. In one embodiment, these molecules interfere with prion formation and/or replication, e.g. by preventing interactions of proteins involved in a prion complex or by interfering with xcex2-sheet formation. In another embodiment, the molecules of the invention promote PrPC conversion to PrPSc, e.g. by binding to PrPC and facilitating a conformational change from PrPC to PrPSc. The molecules may be designed to be species specific, meaning that the molecule will only bind to PrPC or Prion Protein Modulator Factor (PPMF) of the same or a genetically similar species. Alternatively, the molecules of the invention may be designed to bind to PrPC or PPMF of genetically a diverse species, i.e. the molecules will not be limited by the xe2x80x9cspecies barrierxe2x80x9d that normally limits prion infectivity.
The invention features a pharmacophore (defined here as a compound corresponding to a geometric and chemical description of a molecular structure or collection of molecular structures) characterized by an ability to modulate conversion of PrPC to PrPSc in vivo. The pharmacophore can be a peptide or a small molecule with the ability to bind to PPMF and/or PrPC. The structure of the pharmacophore can be defined by a tertiary surface reflecting the negative image of PPMF at its PrP binding domain and/or a tertiary surface defined by the positive image of a specific discontinuous epitope of PrP protein that includes a small subset of residues.
In a preferred embodiment, the pharmacophore structure reflects geometric and chemical positions defined by the relative positions of specific amino acid side chains corresponding to the positions of residues 90-231 of the human PrP protein, and in particular residues 168, 172, 215 and 219 corresponding to the human PrP protein. Optionally or alternatively, the pharmacophore can also contain an epitope from PPMF that binds to PrP.
An object of the invention is to provide an ex vivo system for studying the structural events occurring in conversion, where the system is a cell line treated with a small organic molecule or a peptide that is able to mimic the chemical and geometric features of proteins involved in prion complexing.
An advantage of the present invention is that infectivity of prions in a sample can be determined rapidly.
In another aspect of the invention, the pharmacophore is any one of a collection of molecules that repress prion infectivity and or progression of prion-mediated disease. Any pharmacophore of the invention may inhibit initial infectivity, conversion of PrPC to PrPSc and/or progression of neurodegeneration by any number of mechanisms, including but not limited to binding a molecule involved in prion complexing, e.g. PrPC or PPMF or inhibiting xcex2-sheet formation or elongation.
Yet another aspect of the invention features a method of repressing conversion of PrPC to PrPSc, comprising administering an inhibitor that meets the criteria specified by the pharmacophore model. This may be administered prophylactically to a subject at risk of developing a prion-mediated disorder, e.g. a mammal exposed to infectious prions, or to treat a subject that is exhibiting signs of prion-mediated neurodegeneration.
A feature of the invention is that the inhibitors can be used to treat subjects suffering from prion-mediated disorders.
Yet another aspect of the invention features an assay to identify a PrP pharmacophore, a geometric and chemical specification of a collection of small molecules that could inhibit PrPSc formation. The assay utilizes the steps of determining functional residues of the PrP protein involved in prion complex interactions, developing three dimensional structures based on these functional residues, comparing the three dimensional structures with a series of compounds having known or calculated tertiary structures, and identifying compounds having a spatial orientation consistent with binding to components of the PrPSc replication complex (PrPC, PrPSc, PPMF) at these functional residues.
These and other objects, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the molecules as more fully described below.